Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders. Parkinson's disease (PD) is characterized by the progressive degeneration of dopamine (DA) neurons projecting from the substantia nigra pars compacta (SNpc) to the dorsal striatum. The resulting loss of dopamine in the striatum leads to debilitating motor dysfunction, including rigidity, resting tremor, postural instability, and bradykinesia. Familial or genetic causes of PD only account for 10% of all cases, whereas 90% are considered sporadic and may manifest as a result of a variety of factors. Currently available therapies for Parkinson's disease (PD) provide relief of motor symptoms, but fall short of exhibiting the neuroprotective effect required to prevent progressive degeneration of dopamine (DA) neurons. Furthermore, no therapies simultaneously target the multiple, debilitating, non-motor symptoms of PD.
Alzheimer's disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by loss of memory and cognitive deficits, strongly influenced by the metabolic status, in which the impairment of neuropeptides/neurotransmitters systems has been previously observed. Currently available therapies for Alzheimer's disease (AD) have shown limited efficacy, with no true cure to this day being present. Recent work, both clinical and experimental, indicates that many neurodegenerative disorders often display a coexisting metabolic dysfunction which may exacerbate neurological symptoms. It stands to reason therefore that metabolic pathways may themselves contain promising therapeutic targets for major neurodegenerative diseases.
Ghrelin is a bioactive peptide that induces food intake, body weight gain, and adiposity in rodents (Tschop M. et al., Nature 407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47 (2001)). Acute administration of ghrelin induces food intake in healthy men and women (Wren A. M. et al., J. Endocrinol. Metab. 86:5992-95 (2001): Druce M. R. et al., Int. J. Obes. Relat. Metab. Disord. 29:1130-36 (2005)) as well as in cancer patients with anorexia (Neary N. M. et al., J. Clin. Endocrinol. Metab. 89:2832-36 (2004)). Repeated administration of ghrelin increases lean body mass, body weight and food intake in cachectic patients with Chronic Obstructive Pulmonary Disease (COPD) (Nagaya N. et al., Chest 128:1187-93 (2005)) and improved muscle wasting in patients with chronic heart failure (Nagaya N. et al., Circulation 110:3674-79 (2004)). A similar effect was also shown in a mouse model (Hanada T. et al., Biochem. Biophys. Res. Commun. 301:275-79 (2003)).
Ghrelin has been associated with the progression of obesity and metabolic syndrome, but has been also linked to neuromodulation, neuroprotection and memory and learning processes. In addition, ghrelin system also acts in an autocrine/paracrine fashion where the majority of its components ghrelin variants, acylation enzyme (GOAT) and receptors (GHS-Rs)] are expressed in the different regions of central nervous system. Gahete et al. analyzed the mRNA expression of the ghrelin system in three different regions of the temporal gyros (inferior, medial and superior) of control and AD human brains, since the temporal lobe is considered as one of the most important cortical structures in memory and cognition, and is one of the most affected regions in AD (Gahete et al., Peptides 32: 2225-2228 (2011)). This report showed, for the first time, that AD patients have a reduction in local brain ghrelin production, as compared with age-matched controls. In addition, they analyzed the expression of a newly described In1-ghrelin splice variant (In1-ghrelin is identical to applicants SEQ ID 32 from U.S. Pat. No. 7,176,292), which could also be acylated by the ghrelin-O-acyltransferase (GOAT) enzyme. Results revealed that, similar to ghrelin, In1-ghrelin variant (In1-ghrelin is applicants SEQ ID 32 from U.S. Pat. No. 7,176,292) is also expressed in the temporal lobe, and is down-regulated in AD, thereby showing, together with ghrelin, a region-dependent alteration with AD. Consistent with a similar distribution between ghrelin and GOAT in other tissues, GOAT was also expressed in the temporal lobe of the brain and was impaired in AD, suggesting that a functional autocrine and/or paracrine pathway might be in place within the temporal lobe, and that changes in locally produced acylated/non-acylated ghrelin and In1-ghrelin variant may be of (paths)-physiologically relevance.
The first evidence showing a direct effect of ghrelin on AD-like alterations was reported in a mouse model widely used to examine the pathophysiology of early defects seen in AD. The senescence-accelerated mouse prone8 or SAMP8 mice develop early abnormalities in learning and memory related to abnormalities in septa-hippocampal function, which are due to overproduction of β-amyloid. In this mouse model, ghrelin was able to improve retention of T-maze foot shock avoidance in 12 and 14 month-old mice, compared to their controls (Diano et al. Nat Neurosci. 9:381-8 (2006)). More recently, a different mouse model has been used to analyze in more detail the role that ghrelin plays in AD-related endpoints. This model was generated by intrahippocampal injection of oligomeric forms of the Aβ peptide (AβO), which have been directly related with AD-associated damage (Moon et al. J. Alzheimers Dis.; 23:1.47-59 (2011)). Results of this study revealed that systemic injection of ghrelin rescues memory deficits observed following intrahippocampal AβO injection, using two independent behavioral paradigms (Y-maze and passive avoidance tasks). In addition, the AD-associated neuropathological abnormalities observed in these AβO mice were also attenuated by ghrelin. Indeed, ghrelin inhibited the reactive microgliosis originated by AβO, thus preventing the inflammatory response. Ghrelin also prevented AβO-induced neuronal cell loss in the dentate gyrus and increased the density of hippocampal synaptic and cholinergic nerve fibers. Collectively, these data show that systemic injection of ghrelin rescue cognitive impairments induced by AβO, possibly through inhibition of both, microgliosis and impairment of neuronal integrity.
Recent data (Andrews et al. The Journal of Neuroscience 29(45):14057-14065 (2009)) demonstrate that ghrelin administration decreases substantia nigra pars compacta DA, cell loss and limits striatal DA loss in a PD mouse model. Ghrelin has also been shown to defend against depression (Lutter et al. Nature Neuroscience 11(7):752-3 (2008)) and to stimulate appetite (Tschop M. et al., Nature 407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47 (2001)) in animal models, suggesting additional benefit to these distressing non-motor symptoms of PD (Uc et al. Movement Disorders 21(7):930-6 (2006); Chen et al. Annual Neurology 53(5):676-9 (2003); Delinkanaki-Skaribas et al. Movement Disorders 15; 24(5):667-71 (2009)). It was also shown that subjects at putative preclinical (iRBD) and clinical stages of PD suffer from an impaired ghrelin excretion. Lately it was shown that the GHSR1 and DRD2 receptors form heteromers in hypothalamic neurons and that you can inhibit dopamine signaling using GHSR1 antagonists (Kern A. et al., Neuron 73:317-332 (2012)). From the studies conducted, ghrelin has an acute effect on the firing rate of SNpc DA neurons, which enhances dopamine availability during the course of degeneration and lowers the loss of dopamine levels in the dorsal striatum. In addition, ghrelin-induced enhancement of UCP2-dependent mitochondrial respiration and proliferation provides a bioenergetic status that makes these neurons more resistant to cellular stress. UCP2 is a mitochondrial protein known to protect SNpc DA cells from MPTP intoxication5. Ghrelin also regulates UCP2 mRNA in the brain, consistent with the role of UCP2 as a component of ghrelin-induced neuroprotection. Ghrelin−/− and ghsr−/− mice were more susceptible to DA cell loss in the SNpc and DA loss in the striatum after MPTP than their wt littermates. This suggests that any factor leading to reduced ghrelin production or secretion, whether genetic or environmental, may predispose individuals to nigrostriatal dopaminergic dysfunction. Taken together, these data provide a strong biological rationale for ghrelin peptide family as a novel class of neurodegenerative diseases therapeutics that promotes neuroprotection and relieves non-motor symptoms.
The GHRL (ghrelin) gene encodes a variety of products resulting from alternatively spliced transcripts, various types of cleavage of the prepropeptide, and various post-translational modifications (Kojima M. & Kangawa M., Physiol. Rev. 85:495-522 (2005); Zhang J. V. et al., Science 310:996-99 (2005)). In addition, different degradation products are produced by various tissues (De Vriese C. et al., Endocrinology 145:4997-5005 (2004)). Some of these GHRL products are described herein.
Ghrelin is a 28 amino acid peptide bearing an n-octanoyl side chain on the third serine, resulting from the cleavage of signal and propeptide from the 117 amino acid preproghrelin and an acylation event. The acylated N-terminus of ghrelin is essential for the endocrine functions (Kojima M. et al., Nature 402:656-60 (1999); Bednarek M. A. et al., J. Med. Chem. 43:4370-76 (2000)). Des-acyl ghrelin, which lacks the endocrine functions, was shown to have an antagonistic effect to that of ghrelin on glucose output in vitro (Gauna C. et al., J. Clin. Endocrinol. Metab. 89:5035-42 (2004)). An alternatively-spliced ghrelin mRNA encodes a 116 amino acid prepropeptide that is further processed to a Des-Gln14-ghrelin and a 27 amino acid processed peptide (Hosoda H. et al., J Biol. Chem. 275:21995-22000 (2000)). Another peptide, Obestatin, is cleaved from the preproghrelin and has no sequence overlap with processed ghrelin peptide. This peptide was shown to have some antagonistic effect to acylated ghrelin, inhibiting food intake and body weight gain (Zhang J. V. et al., Science 310:996-99 (2005)). Yet another peptide, the 66 amino acid C-terminus of the preproghrelin, may also be functional (Pemberton C. et al., Biochem. Biophys. Res. Comm. 310:567-73 (2003)). A variety of isoforms, including isoforms encoded by different splice variants, are known for other proteins, e.g. for vascular endothelial growth factor (VEGF), where different isoforms share roles as angiogenesis, while differing in some other characteristics, such as binding affinity (Neufeld G. et al., FASEB J. 13:9-22 1999). Thus, the variety of products of the GHRL gene may reflect a similarly complex control of the endocrine and paracrine action of the ghrelin isoforms.
Previously, administration of ghrelin by continuous infusions of 5 pmol/kg/min doses for 270 minutes was shown to increase food intake in healthy humans (Wren A. M. et al., J. Clin. Endocrinol. Metab. 86:5992-95 (2001)). It was also shown that infusion of ghrelin for 90 minutes could increase food intake by 30% in cancer cachexia patients (Abstract P09, Digestive Hormones, Appetite and Energy Balance, Baylis and Starling meeting, London, June 2003). Recently, it was shown that subcutaneous injection of 3.6 nmol/kg acylated ghrelin prior to a meal, thereby ensuring a close mimic of the natural pre-meal situation, increased energy intake by 27%. Ghrelin also appeared to enhance the perceived palatability of the food offered (Druce M. R. et al., Int. J. Obes. Relat. Metab. Disord. 29:1130-36 (2005)).
These studies demonstrate that parenteral administration of ghrelin can increase appetite in both normal subjects and in patients with loss of appetite. Furthermore, Applicant has found that it is possible to obtain a significant effect of body weight gain and a significant increase in food consumption with a novel ghrelin splice variant (see co-owned, published U.S. Pat. No. 7,981,860 and/or U.S. Pat. No. 7,763,707, incorporated herein by reference) when administered to a subject, in particular when administered subcutaneously prior to a meal, thereby ensuring a close mimic of the natural pre-meal situation. Applicant's novel ghrelin splice variant effect of weight gain is mainly on lean mass mass while ghrelin's effect on weight gain is mainly on fat mass.
In addition applicant has found that it is possible to decrease substantia nigra pars compacta DA cell loss and limits striatal DA loss with a novel ghrelin splice variant (see co-owned, published U.S. Pat. No. 7,981,860 and/or U.S. Pat. No. 7,763,707, incorporated herein by reference) when administered to a subject in a PD animal model.